Tubulin Polymerization Inhibitors Research Articles

Red‐Light Activation of a Microtubule Polymerization Inhibitor via Amide Functionalization of the Ruthenium Photocage

AbstractPhotoactivated chemotherapy (PACT) is a promising cancer treatment modality that kills cancer cells via photochemical uncaging of a cytotoxic drug. Most ruthenium‐based photocages used for PACT are activated with blue or green light, which penetrates sub‐optimally into tumor tissues. Here, we report amide functionalization as a tool to fine‐tune the toxicity and excited states of a terpyridine‐based ruthenium photocage. Due to conjugation of the amide group with the terpyridine π system in the excited state, the absorption of red light (630 nm) increased 8‐fold, and the photosubstitution rate rose 5‐fold. In vitro, red light activation triggered inhibition of tubulin polymerization, which led to apoptotic cell death both in normoxic (21 % O2) and hypoxic (1 % O2) cancer cells. In vivo, red light irradiation of tumor‐bearing mice demonstrated significant tumor volume reduction (45 %) with improved biosafety, thereby demonstrating the clinical potential of this compound.

A novel pyrrolidine-2,5-dione derivative induced G2/M phase arrest and apoptosis of hepatocellular carcinoma HepG2 cells through inhibiting tubulin polymerization

Hepatocellular carcinoma (HCC) is a major global cause of carcinoma-related fatality. The inhibition of tubulin polymerization holds significant potential in the development of cancer drugs. In our study, we synthesized a novel pyrrolidine-2,5-dione derivative (compound 8) that exhibited potent anti-HCC activity (IC50 value of 2.082 μM) against human HCC HepG2 cells. Moreover, compound 8 significantly suppressed the HepG2 cell multiplication, and triggered G2/M phase arrest of HepG2 cells and their apoptosis. Further mechanistic investigations revealed that compound 8 suppressed tubulin polymerization by directly binding to the colchicine binding site of β-tubulin. Additionally, compound 8 significantly inhibited tumor growth with low toxicity in nude HepG2 tumor-bearing mice, achieving an approximate inhibition rate of 45.73 %. Therefore, compound 8 represents a promising pharmaceutical candidate for HCC management.

3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines inhibit tubulin polymerisation and act as anticancer agents

A series of cis-restricted 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines as novel tubulin polymerisation inhibitors was designed based on molecular docking. Compound 9p, exhibited potent antiproliferative activity against HeLa, MCF-7, and A549 cell lines. Mechanism studies indicated that 9p potently inhibited tubulin polymerisation and disrupted the microtubule dynamics of tubulin in HeLa cells. Moreover, 9p could cause G2/M phase cell cycle arrest and apoptosis in HeLa cells. In addition, the prediction of physicochemical properties disclosed that 9p conformed well to the Lipinski’s rule of five. The initial results suggest that the 3-aryl-4-(3,4,5-trimethoxyphenyl)pyridines could serve as a promising scaffold for the development of novel anticancer drugs.

Natural-Product-Inspired Discovery of Trimethoxyphenyl-1,2,4-triazolosulfonamides as Potent Tubulin Polymerization Inhibitors.

An approach of natural product-inspired strategy and incorporation of an NP-privileged motif has been investigated for the discovery of new tubulin polymerization inhibitors. Two series, N-Arylsulfonyl-3-arylamino-5-amino-1,2,4-triazole derivatives, and their isomers were considered. The compounds were synthesized by construction of the N-aryl-1,2,4-triazole-3,5-diamine motif and sulfonylation. Although the chemo- and regioselectivity in sulfonylation were challenging due to multiple ring-tautomerizable-NH and exocyclic NH2 functionalities present in the molecular motifs, the developed synthetic method enabled the preparation of designed molecular skeletons with biologically important motifs. The approach also led to explore interesting molecular regio- and stereochemical aspects valuable for activity. The X-ray crystallography study indicated that the hydrogen bonding between the arylamine-NH and the arylsulfonyl-"O" unit and appropriate molecular-functionality topology allowed the cis-locking of two aryls, which is important for tubulin-binding and antiproliferative properties. All synthesized compounds majorly showed characteristic antiproliferative effects in breast cancer cells (MCF-7), and four compounds exhibited potent antiproliferative activity. One compound potently bound to tubulin at the colchicine site and inhibited tubulin polymerization in vitro. The compound significantly depolymerized microtubules in MCF-7 cells, arrested the cells at the G2/M phase, and induced cell death. This study represents the importance of the design strategy in medicinal chemistry and the molecular structural features relevant to anticancer anti-tubulin properties. The explored molecules have the potential for further development.

Identification of acetylshikonin as a novel tubulin polymerization inhibitor with antitumor activity in human hepatocellular carcinoma cells.

Microtubules are attractive targets for anticancer drugs. However, the microtubule-targeting agents (MTAs) currently in clinical use exhibit inevitable drug resistance. Therefore, there is an urgent need to discover novel MTAs for the clinical treatment of cancer. Bioactive compounds extracted from Lithospermum erythrorhizon were assessed for in vitro anti-proliferative activities against a panel of human cancer cell lines using cell counting kit-8 (CCK-8) assay. Tubulin polymerization inhibition assay, colchicine competitive binding site assay, and immunofluorescence were used to validate the tubulin inhibition effect of acetylshikonin. Flow cytometry, Hoechst staining, and caspase-3 activity evaluation were performed to assess cell cycle arrest and cell apoptosis. 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide (JC-1) staining and dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining were used to evaluate mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. Acetylshikonin exhibited potent anti-proliferative activities against a panel of human cancer cell lines (IC50 values: 1.09-7.26 µM) and displayed comparable cytotoxicity against several drug-resistant cell lines. Further mechanism studies revealed that acetylshikonin induced cell cycle arrest of MHCC-97H cells at G2/M phase, and significantly promoted apoptosis marked by a collapse of MMP and abnormal ROS accumulation. In this study, acetylshikonin was identified as MTA against hepatocellular carcinoma and can serve as a promising lead compound for further development of anti-cancer drug, underscoring its potential clinical significance.

Abstract A027: A photoactivatable microtubule-targeting rigidin prodrug

Abstract Analogues of marine alkaloid rigidins have been found to kill cancer cells at nanomolar concentrations by targeting the microtubule network. Here, a rigidin analogue containing a thioether group was “caged” by coordination of its thioether group to a photosensitive ruthenium polypyridyl complex. In the dark, the coordinated ruthenium fragment prevented the rigidin analogue from inhibiting tubulin polymerization and reduced its toxicity in 2D cancer cell lines monolayers, 3D lung cancer tumor spheroids (A549), and in a lung cancer tumor xenograft (A549) in nude mice. Photochemical activation of the prodrug upon green light irradiation led to the photosubstitution of the thioether ligand by water, thereby releasing the free rigidin analogue capable of inhibiting the polymerization of tubulin. In cancer cells, such photorelease was accompanied by a drastic reduction of cell growth, not only when the cells were grown in normoxia (21% O2) but also, remarkably, in hypoxic conditions (1% O2). In vivo, light activation of the compound in the tumor led to 30% tumor volume reduction, which represents the first demonstration of the efficacy of a ruthenium-based photocaged compounds in a tumor xenograft. In subsequent unpublished work, we fine-tuned the excited states in this complex by adding an amide functional group on the ruthenium cage. By doing so, a new caged complex was obtained that was activated by red light (instead of green light). We demonstrated the red light activation of this compound in solution and in vitro. This result demonstrates that functionalization of ruthenium cage with electron-withdrawing groups can radically enhance their photochemical properties, which is essential for this method development (red light penetrates better into tissues than green light). We will present our results of in vivo testing of this caged compound activated by tumor penetrable red light, where significant tumor reduction is seen in mice xenografted with subcutaneous human uveal melanoma liver metastatic cells. Citation Format: Alexander Kornienko, Sylvestre Bonnet. A photoactivatable microtubule-targeting rigidin prodrug [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A027.

Design, Synthesis, in Silico and in Vitro Evaluation of New Combretastatin A-4 Analogs as Antimitotic Antitumor Agents.

Combretastatin A-4 (CA-4) binds β-tubulin at the colchicine-binding site preventing tubulin from polymerizing into microtubules. CA-4 and cis combretastatin analogs isomerize to the trans form resulting in decreased cytotoxicity and anti-tubulin activity. However, the excellent anti-cancer potential and relatively simple molecular structure of CA-4, provide an encouraging starting point for the development of new, more stable and more potent anti-tubulin compounds. To synthesize a new series of 4-(3,4,5-trimethoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione derivatives (compounds 10-12) as analogs of CA-4, with different alkyl, and aryl side chain substituents at the triazole moiety (B-ring), resulting in permanent cis form. The newly synthesized CA-4 analogs were characterized by FT-IR, 1HNMR, 13CNMR, and HR-MS(ESI) techniques. Molecular docking studies including docking score (G) ADMET, DFT, and mlocular similarities were performed. The anti-proliferative activity of the new compounds against three human cancer cell lines was evaluated using the MTT assay, and their ability to inhibit tubulin polymerization, and consequently their effects on cell cycle progression and induction of apoptois were assessed. Molecular docking studies showed that compounds 11b and 11d exhibited the highest docking scores (-13.30 and -14.01 Kcal/mol, respectively) into colchicine-binding site, scores very close to the reference drug colchicine (-13.50 Kcal/mol), and that hydrogen bonding and hydrophobic interaction are essential for binding. The most active cytotoxic compound 11b had potent IC50 values ranging from 3.83 to 10.67 µM against the three human cancer cell lines, while exhibiting low cytotoxicity against non-cancer human WI-38, suggesting that compound 11b targets rapidly growing cancer cells. Moreover, compound 11b exhibited potent anti-tubulin activity which was comparable to CA-4. Targeting microtubules caused cell cycle arrest at the G2/M phase resulting in induction of apoptosis. These findings indicate that compound 11b is a promising β-tubulin-binding compound with antimitotic action that has the potential to treat cancer.

Discovery of a Dual Tubulin and Neuropilin-1 (NRP1) Inhibitor with Potent In Vivo Anti-Tumor Activity via Pharmacophore-based Docking Screening, Structure Optimization, and Biological Evaluation.

Dual inhibition of tubulin and neuropilin-1 (NRP1) may become an effective method for cancer treatment by simultaneously killing tumor cells and inhibiting tumor angiogenesis. Herein, we identified dual tubulin/NRP1-targeting inhibitor TN-2, which exhibited good inhibitory activity against both tubulin polymerization (IC50 = 0.71 ± 0.03 μM) and NRP1 (IC50 = 0.85 ± 0.04 μM). Importantly, it significantly inhibited the viability of several human prostate tumor cell lines. Further mechanism studies indicated that TN-2 could inhibit tubulin polymerization and cause G2/M arrest, thereby inducing cell apoptosis. It could also suppress cell tube formation, migration, and invasion. Moreover, TN-2 showed obvious antitumor effects on the PC-3 cell-derived xenograft model with negligible side effects and good pharmacokinetic profiles. These data demonstrate that TN-2 could be a promising dual-target chemotherapeutic agent for the treatment of prostate cancer.

RBIO-05. MITOTIC ENRICHMENT AS AN EFFICIENT RADIOSENSITIZATION STRATEGY FOR INTRACRANIAL TUMORS

Abstract Radiotherapy remains one of the most effective modalities for anticancer treatment and part of standard of care for many primary and metastatic brain tumors. Boosting the efficacy of radiotherapy is therefore a logical avenue to improve patient survival. We have developed a radiosensitization strategy called ‘induction of mitotic enrichment’. It has long been known that the radiosensitivity of a cell depends on the phase of the cell cycle and that mitotic cells are especially vulnerable. Enriching the tumor for mitotic cells by arresting them during division prior to each radiotherapy fraction should therefore render the tumor population more sensitive to irradiation. Ideally, induction of mitotic enrichment should be reversible and non-cytotoxic to prevent healthy tissue toxicity and be compatible with clinically applied fractionated radiotherapy regimens. We have now identified an orally available targeted tubulin polymerization inhibitor that can achieve repeated and reversible mitotic enrichment for up to 10 hours prior to radiotherapy, without causing cytotoxicity in vitro or healthy tissue toxicity in vivo. This tubulin inhibitor (ABT-751) efficiently radiosensitizes a range of preclinical tumor models representing adult and pediatric, primary and metastatic brain tumors. Importantly, ABT-751 also improves survival in mouse models of adult glioblastoma (GBM), but only in a mitotic enrichment setup when given several hours prior to radiotherapy to allow accumulation in mitosis. Of note, induction of mitotic enrichment does not exacerbate healthy tissue toxicity. To conclude, induction of mitotic enrichment by ABT-751 radiosensitizes adult GBM to fractionated radiotherapy. We are currently expanding our preclinical development of mitotic enrichment as a radiosensitization strategy to other targets and cancers and have designed a phase 0/I trial for adult GBM to demonstrate induction of mitotic enrichment and tolerability when combined with radiotherapy. We expect to start enrollment in this trial soon.

Structure-Based Optimization of Carbendazim-Derived Tubulin Polymerization Inhibitors through Alchemical Free Energy Calculations.

Carbendazim derivatives, commonly used as antiparasitic drugs, have shown potential as anticancer agents due to their ability to induce cell cycle arrest and apoptosis in human cancer cells by inhibiting tubulin polymerization. Crystallographic structures of α/β-tubulin multimers complexed with nocodazole and mebendazole, two carbendazim derivatives with potent anticancer activity, highlighted the possibility of designing compounds that occupy both benzimidazole- and colchicine-binding sites. In addition, previous studies have demonstrated that the incorporation of a phenoxy group at position 5/6 of carbendazim increases the antiproliferative activity in cancer cell lines. Despite the significant progress made in identifying new tubulin-targeting anticancer compounds, further modifications are needed to enhance their potency and safety. In this study, we explored the impact of modifying the phenoxy substitution pattern on antiproliferative activity. Alchemical free energy calculations were used to predict the binding free energy difference upon ligand modification and define the most viable path for structure optimization. Based on these calculations, seven compounds were synthesized and evaluated against lung and colon cancer cell lines. Our results showed that compound 5a, which incorporates an α-naphthyloxy substitution, exhibits the highest antiproliferative activity against both cancer lines (SK-LU-1 and SW620, IC50 < 100 nM) and induces morphological changes in the cells associated with mitotic arrest and mitotic catastrophe. Nevertheless, the tubulin polymerization assay showed that 5a has a lower inhibitory potency than nocodazole. Molecular dynamics simulations suggested that this low antitubulin activity could be associated with the loss of the key H-bond interaction with V236. This study provides insights into the design of novel carbendazim derivatives with anticancer activity.

Design, synthesis and bioevaluation of novel trifluoromethylquinoline derivatives as tubulin polymerization inhibitors.

Aim: A series of novel trifluoromethylquinoline derivatives were designed, synthesized and evaluated for antitumor activities. Methodology: All compounds were evaluated for antiproliferative activity against four human cancer cell lines. Results: Among them, 5a, 5m, 5o and 6b exhibited remarkable antiproliferative activities against all the tested cell lines at nanomolar concentrations. Mechanism of action studies demonstrated that 6b targeted the colchicine binding site, potentially inhibiting tubulin polymerization, and further studies indicated that 6b could arrest LNCaP cells in the G2/M phase and induce cell apoptosis. Molecular docking confirmed that 6b could bind to the colchicine binding site. Conclusion: Results suggested that 6b could serve as a promising lead compound for the development of novel tubulin polymerization inhibitors and cancer therapy.

A Concise Review of Synthetic Strategy, Mechanism of Action, and SAR Studies of Phthalazine Derivatives as Anticancer Agent

Background: Colorectal cancer is the third foremost cause of death in women and men. Globally, about 1.94 million colon cancer cases were diagnosed and around 0.93 million patients died in the previous year. Introduction: Several drugs have been permitted by the Food And Drug Administration (FDA) for the treatment of colorectal cancer. The main difficulties of current drugs are the expansion of resistance issues, target selectivity issues and toxicity issues. The existing therapies, such as surgery and hormonal therapy, are in use but exhibit numerous adverse effects, such as pharmacokinetic issues and pharmacodynamic issues. Hence, hereby is a crucial requirement of novel moieties that are peaceable and efficient in the handling of colorectal cancer. Method: Phthalazine derivatives have expanded admiration over a few years due to their efficient anticancer significance. These Phthalazine derivatives exhibit anticancer activity by targeting various mechanisms such as apoptosis induction, tubulin polymerization inhibition, EGFR inhibition, and aurora kinase inhibition. Result: In this study, we have focused on the Structural Activity relationship, numerous synthetic strategies and mechanism of action of phthalazine derivatives for potential treatment of cancer. Conclusion: Among some of phthalazine derivative compounds not only induced antiproliferative activity even also improved bioavailability and reduced side effects, like 4-(phthalazine-1-yl) aniline with (IC50 = 0.22 ± 0.11 μM), and 4-phthalazin-1-yl-amino) benzonitrile (IC50 = 1.20 μM), 4-((5- methyl-pyrazole-3-yl) amino)-2-phenylphthalazin-1-one (IC50 = 0.031 μM) and 4-((5-methylpyrazole- 3-yl) amino)-2-(p-tolyl)phthalazin-1-one (IC50 = 0.065 μM). Therefore, this study would be the inspiration for the betterment of human health.

Probing a distinct druggable tubulin binding site with gatorbulins 1–7, their metabolic and physicochemical properties, and pharmacological consequences

Microtubules, consisting of α/β-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2–7 (1b–1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and β-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

Verubulin (Azixa) Analogues with Increased Saturation: Synthesis, SAR and Encapsulation in Biocompatible Nanocontainers Based on Ca2+ or Mg2+ Cross-Linked Alginate.

Tubulin-targeting agents attract undiminished attention as promising compounds for the design of anti-cancer drugs. Verubulin is a potent tubulin polymerization inhibitor, binding to colchicine-binding sites. In the present work, a series of verubulin analogues containing a cyclohexane or cycloheptane ring 1,2-annulated with pyrimidine moiety and various substituents in positions 2 and 4 of pyrimidine were obtained and their cytotoxicity towards cancer and non-cancerous cell lines was estimated. The investigated compounds revealed activity against various cancer cell lines with IC50 down to 1-4 nM. According to fluorescent microscopy data, compounds that showed cytotoxicity in the MTT test disrupt the normal cytoskeleton of the cell in a pattern similar to that for combretastatin A-4. The hit compound (N-(4-methoxyphenyl)-N,2-dimethyl-5,6,7,8-tetrahydroquinazolin-4-amine) was encapsulated in biocompatible nanocontainers based on Ca2+ or Mg2+ cross-linked alginate and it was demonstrated that its cytotoxic activity was preserved after encapsulation.

Design, Synthesis, and Antitumor Efficacy of Substituted 2-Amino[1,2,4]triazolopyrimidines and Related Heterocycles as Dual Inhibitors for Microtubule Polymerization and Janus Kinase 2.

Preclinical and clinical studies have demonstrated the synergistic effect of microtubule-targeting agents in combination with Janus kinase 2 (JAK2) inhibitors, prompting the development of single agents with enhanced therapeutic efficacy by dually inhibiting tubulin polymerization and JAK2. Herein, we designed and synthesized a series of substituted 2-amino[1,2,4]triazolopyrimidines and related heterocycles as dual inhibitors for tubulin polymerization and JAK2. Most of these compounds exhibited potent antiproliferative activity against the selected cancer cells, with compound 7g being the most active. This compound effectively inhibits both tubulin assembly and JAK2 activity. Furthermore, phosphorylated compound 7g (i.e., compound 7g-P) could efficiently convert to compound 7g in vivo. Compound 7g, whether it was administered directly or in the form of a phosphorylated prodrug (i.e., compound 7g-P), significantly inhibited the growth of A549 xenografts in nude mice. The present findings strongly suggest that compound 7g represents a promising chemotherapeutic agent with high antitumor efficacy.

Design, synthesis and biological evaluation of N-benzylaryl cinnamide derivatives as tubulin polymerization inhibitors capable of promoting YAP degradation with potent anti-gastric cancer activities

In this work, we utilized the N-benzylaryl derivative 9 as a lead compound and employed the molecular hybridization strategy by introducing cinnamoyl fragments to successfully design and synthesize 33 novel N-benzylaryl cinnamide derivatives 15a∼15 ag. The in vitro antiproliferative activities were explored, and the preliminary analysis and summary of their structure-activity relationship were conducted. The majority of the compounds demonstrated significant inhibitory potency on MGC-803, HCT-116 and KYSE450 cells with IC50 values below 0.5 μM. Among them, compound 15e (MY-1076) exhibited the most effective effect on the proliferative inhibition of MGC-803, SGC-7901, HCT-116 and KYSE450 cells with IC50 values of 0.019, 0.017, 0.020 and 0.044 μM, respectively, which is more potent than colchicine and the lead compound 9. Additionally, compound 15e (MY-1076) still exhibited significant inhibitory proliferation activity against 13 other types of tumor cells (IC50 values < 0.1 μM). Further studies revealed that compound 15e (MY-1076) could effectively inhibit tubulin polymerization by acting on the β-tubulin colchicine binding site, thereby disrupting microtubule network assembly and mitotic progression. Additionally, compound 15e (MY-1076) also demonstrated a notable inhibitory effect on the oncogenic protein YAP by inducing its degradation. Compound 15e (MY-1076) could dose-dependently induce G2/M phase arrest and cell apoptosis, effectively inhibit the colony formatting ability and cause morphological changes in MGC-803 and SGC-7901 cells. Compound 15e (MY-1076) exhibited significant regulatory effects on the expression levels of cell cycle and apoptosis-related proteins. Taken together, we here reported a novel N-benzylaryl cinnamide derivative 15e (MY-1076) as a tubulin polymerization inhibitor capable of promoting degradation of YAP, which held great potential as an anti-gastric cancer agent.

Design, synthesis and biological evaluation of novel dihydroquinolin-4(1H)-one derivatives as novel tubulin polymerization inhibitors

A series of novel dihydroquinolin-4(1H)-one derivatives targeting colchicine binding site on tubulin were designed, synthesized and evaluated as anticancer agents. The most potent compound 6t showed remarkable antiproliferative activities against four cancer cell lines with IC50 values among 0.003–0.024 μM and tubulin polymerization inhibitory activity (IC50 = 3.06 μM). Further mechanism studies revealed that compound 6t could induce K562 cells apoptosis and arrest at the G2/M phase. Meanwhile, 6t significantly inhibited migration and invasion of MDA-MB-231 cells, and disrupted the angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro. In addition, compound 6t inhibited tumor growth in H22 allograft tumor model with a tumor growth inhibition (TGI) rate of 63.3 % (i.v., 20 mg/kg per day) without obvious toxicity. Collectively, these results indicated that compound 6t was a novel tubulin polymerization inhibitor with potent anticancer properties in vitro and in vivo.

Mechanisms of cytotoxic activity of pyrrole-carboxamides against multidrug-resistant tumor cell sublines

Introduction. Mitotic poisoning agents (MPAs) affecting the dynamic state of the microtubules, are the well-known and effective chemotherapeutic agents. Mitotic poisoning agents are binding to the microtubules, and thereby interfere with tubulin polymerization or depolymerization dynamic state, resulting in the cell cycle arrest in M-phase (mitotic catastrophe) and subsequent apoptotic cell death. We reported previously about potent cytotoxic activities against the pyrrole-carboxamides (PCs) (PC-61 and PC-84) against broad spectrum of cancer cell lines, including triple negative breast cancer, lung and prostate cancer.Aim. To examine the cytotoxic activities of PC-61 and PC-84 against multidrug-resistant cancer cell lines indicated above.Materials and methods. Studу was performed on the triple-negative paclitaxel-resistant breast cancer cell line HCC1806 Tx-R and doxorubicin-resistant osteosarcoma SaOS-2 Dox-R cell line.Results. The cytotoxic activity of PCs was due to the inhibition of tubulin polymerization. Immunofluorescence staining data revealed PC’s ability to interfere with tubulin’s assembly in multidrug-resistant cancer cell lines. As an outcome of inhibition of tubulin polymerization, PCs induced cell cycle arrest in M-phase, and further led to apoptotic cell death of cancer cells.Conclusion. Collectively, we demonstrated potent cytotoxic activity of PCs against cancer cell lines with multidrug-resistant phenotype, which arising the possibilities to develop novel and effective anti-tumor agents that belongs to mitotic poisoning agents

Benzimidazole derivatives as tubulin polymerization inhibitors: Design, synthesis and in vitro cytotoxicity studies

A new class of benzimidazole derivatives as tubulin polymerization inhibitors has been designed and synthesized in this study. The in vitro anticancer profile of the developed molecules was reconnoitred on selected human cancer cells. The highest cytotoxicity was illustrated by compounds 7n and 7u with IC50 values ranging from 2.55 to 17.89 µM with specificity toward SK-Mel-28 cells. They displayed 5-fold less cytotoxicity towards normal rat kidney epithelial NRK52E cells, which implies that they are not harmful to normal, healthy cells. The cellular staining procedures like AO/EB, DCFDA, and DAPI were applied to comprehend the inherent mechanism of apoptosis which displayed nuclear and morphological alterations. The Annexin V binding and JC-1 studies were executed to evaluate the extent of apoptosis and the decline in mitochondrial transmembrane potential in SK-Mel-28 cell lines. Compound 7n dose-dependently arrested the G2/M phase of the cell cycle and the target-based outcomes proposed tubulin polymerization inhibition by 7n (IC50 of 5.05±0.13 μM). Computational studies were also conducted on the tubulin protein (PDB ID: 3E22) to investigate the stabilized binding interactions of compounds 7n and 7u with tubulin, respectively.

Design, synthesis, docking studies and biological screening of 2-pyrimidinyl-2, 3-dihydro-1H-naphtho [1, 2-e][1, 3] oxazines as potent tubulin polymerization inhibitors

A series of novel substituted 2-pyrimidinyl-2,3-dihydro-1H-naphtho[1,2-e][1, 3]oxazine analogs have been designed and synthesized based on structure-activity relationships from 2-naphthol, substituted pyrimidinyl amines and formalin through ring closure by one-pot three component reaction. These derivatives were evaluated for their in vitro cytotoxicity, cell cycle assay and their inhibitory effect on tubulin polymerization. From the MTT assay, it is clear that most of the synthesized compounds displayed potent cytotoxic activities on HeLa (cervical cancer) and B16F10 (melanoma) cancerous cell lines. The compounds 6b and 6k were found to be more effective against HeLa cell lines and exhibited significant cytotoxicity (with IC50 values 1.26 ± 0.12 µM and 1.16 ± 0.27 µM respectively), accumulation of HeLa cells in G2/M phase and exhibiting induced apoptosis. The immunohistochemistry and fluorescence assays showed that these compounds 6b and 6k inhibited the microtubule assembly in human cervical cancer cells (HeLa) at 2 µM concentration. Furthermore, molecular docking studies of these molecules revealed their better-fit potential as anticancer molecules and have a high affinity for colchicine binding site, indicating more inhibitory potential at the cellular level. Our studies suggest that the newly synthesized compounds may become promising leads for the development of new anti-cancer agents. Communicated by Ramaswamy H. Sarma